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A specific method for the determination of free acetate in blood and tissues

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The amino-terminal sequence of rat liver M4 lactate dehydrogenase has been investigated in an attempt to determine the number of polypeptide chains in this protein. Quantitative dinitrophenylation and phenylthiocarbamylation experiments carried out under a wide variety of conditions yielded only submolar quantities of amino-terminal residues. The conditions used included those which lead to the dissociation of lactate dehydrogenases and those which cause extensive unfolding of the polypeptide chains. The acetylation status of rat liver M4 lactate dehydrogenase was investigated by means of a microenzymatic method developed during this study. The presence of 6 to 8 moles of acetate per mole of rat liver lactate dehydrogenase was shown. Bovine heart H4 and bovine heart MH3 lactate dehydrogenases also contained 7 to 8 moles of acetate per mole. Chicken heart H4 lactate dehydrogenase contained 3 to 4 moles of acetate per mole. All the acetyl groups in rat liver M4 lactate dehydrogenase are N-acetyl residues. Rat liver M4 lactate dehydrogenase also contains a quantity of a carbohydrate-like material which is not easily removed by dialysis, precipitation with ammonium sulfate, or repeated Bio-Gel chromatography. These data, combined with the lack of reactivity of rat liver M4 lactate dehydrogenase toward leucine aminopeptidase (62), lead to the conclusion that the amino-terminal residues of rat liver M4 lactate dehydrogenase are not readily available for reaction with amino-terminal labeling reagents and are probably acetylated. The large number of acetyl residues suggests the presence of more than one polypeptide chain per subunit.
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THE concentration of acetate in the blood determined enzymatically1 in a number of experimental subjects was found to be about 2 µgm./ml., independent of the nutritional régime (see Table 1). Attempts to raise the level of acetate in the blood by oral ingestion of acetic acid or acetates were unsuccessful, because the experimental subjects were not able to consume a sufficient quantity of those substances.
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We infused acetate into normal human subjects and performed kinetic analysis of the plasma and urine values obtained before, during, and after the infusion. The data were best fitted by a first-order elimination process with a mean metabolic clearance rate of 2.3 L/min. Gotch and Sargent had previously suggested that during dialysis, acetate metabolism was zero order. We performed kinetic modeling of acetate concentrations during dialysis. The data were best fitted to a Michaelis-Menton model (i.e., first-order metabolism at low concentrations and saturated at high concentrations). The mean Km for acetate in the dialysis patients was 8.5 mM and the mean Vmax was 18 mmol/min. Patients with a Vmax less than 7 mmol/min usually had a fall in plasma bicarbonate during dialysis while patients with a Vmax greater than 14 mmol/min usually had a rise in bicarbonate during dialysis. It is concluded that during high-surface-area dialysis, the capacity for acetate metabolism will affect acid-base homeostasis. Kinetic modeling will be useful to define acetate-intolerant patients and may be used to predict patients who will benefit from bicarbonate hemodialysis.
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One more case of paraldehyde-induced metabolic acidosis is added to the six previously reported. Observations in dogs suggest that fresh paraldehyde can induce degrees of acidosis comparable to decomposed paraldehyde. The nature of accumulating acid(s) was investigated. At least part appears to arise as the phosphate. The relatively large changes of phosphate may represent acid arising intracellularly.
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Our previous studies suggested that acetic thiokinase may regulate lipogenesis. Therefore, we measured free acetate concentration and observed a significant increase in livers of 72hr fasted rats. Acetyl CoA deacylase activity was also measured in microsomal supernatants and mitochondria. In the latter, it was localized in the inner membrane and the matrix. Fasting did not alter acetyl CoA deacylase activity in either whole mitochondrial extracts or in microsomal supernatants of liver. These results further support the possibility that free acetate is a metabolic intermediate and that increased concentrations of acetate observed in liver of fasted rats may be due to the impaired conversion of acetate to acetyl CoA under conditions of inhibited lipogenesis.
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Methods are presented for the direct spectrophotometric and fluorometric determinations of acetate in tissue extracts using beef heart mitochondria acetyl-CoA synthetase. The methods eliminate the need for diffusion or distillation of the acetate from the extract and thereby minimize the danger of breakdown of labile tissue acetate esters, an occurrence which would result in an overestimation of acetate. Acetyl-CoA, acetylcarnitine, and N-acetyl-aspartate do not break down to acetate during the extraction procedure and do not interfere with the assay.
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Two new cytidine nucleotides have been isolated from Azotobacter vinelandii strain O. One of them has been shown, primarily by application of nuclear magnetic resonance spectroscopy and mass spectrometry, to be cytidine 5'-diphosphate 6-deoxy-3-C-methyl-2-O-methyl-l-aldohexopyranoside.
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Chapter
Mit der Entdeckung des Coenzym A im Jahr 1945 bestätigte sich von neuem die aus anderen Beispielen bekannte Tatsache, daß Vitamine als wesentliche Bausteine von Coenzymen in den geordneten Ablauf von Stoffwechselprozessen eingeschaltet sind. In diesem Falle war es die Pantothensäure, der man endlich einen ihrer vielfältigen Vitaminwirkung angemessenen Platz im Stoffwechselgetriebe zuweisen konnte. Die ungewöhnlich rasche und ergiebige Forschungstätigkeit auf dem Gebiet der Coenzym A-abhängigen Stoffwechselreaktionen ist zweifellos auf den hohen Stand der Methoden der Enzymologie zurückzuführen, in den das neue Coenzym hineingeboren wurde. Als Ergebnis liegen heute viele Dutzende in ihren Eigenschaften und ihrer Wirkungsweise gut bekannte neue Enzyme vor, die uns — von wenigen Ausnahmen abgesehen — ein nahezu lückenloses Bild von der beherrschenden Funktion des Coenzym A im Stoffwechsel vermitteln. Die systematische Abhandlung des Komplexes der Coenzym A-Enzyme darf nunmehr, nachdem die Ernte im wesentlichen eingebracht ist, in Angriff genommen werden.
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The kinetic deuterium isotope effect, D(V/K), on ethanol oxidation was measured by the radiometric, competitive method using 14C-labelled ethanol containing deuterium in the (1-R) position. Acetate was isolated and used for the determination. Experiments were performed on rats either anaesthetized and laparotomized, or provided with indwelling catheters in a. carotis, v. cava and v. portae. Experiments were also made on perfused liver from rats pretreated with acetone, or a mixture of acetone and phenobarbital. Finally, intact non-anaesthetized rabbits were used. The apparent isotope effect in all in vivo experiments decreased rapidly in the presence of acetaldehyde as a consequence of the reversibility of the ADH reaction. In the case of rabbits and catheterized rats this problem was tackled by taking blood samples in quick succession, thus permitting extrapolation of the apparent isotope effect to the time of injection of the labelled ethanol. In anaesthetized rats injection of the ADH inhibitor isobutyramide was used to reduce the concentration of acetaldehyde and thereby the rate of decline of the apparent isotope effect. At high doses of isobutyramide the isotope effect was constant with time at about 1.9 suggesting the presence of non-ADH activity. In all three kinds of in vivo experiments the isotope effect ranged from 2.66 to 2.93. In the case of anaesthetized rats the mean value was 2.89 +/- 0.05 (S.D.). This figure is significantly different from that of rat liver ADH, P less than 0.001. As the figures for the initial isotope effects are minimum values the contribution of non-ADH ethanol oxidizing systems is likely to be small, probably less than 10 percent.
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A simple and rapid method is described for determining the free acetate concentration in liver and serum. After extraction, the acetate is converted to its benzyl ester by thermal degradation of its benzyldimethylphenylammonium salt in the vaporizer of a gas chromatography. Good quantitation is achieved in the range of 0.033-2.5 mumoles of acetate per gram of liver or per milliliter of serum.
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This chapter presents the enzymatic determination of acetate. The assay depends on the conversion of acetate to acetyl-CoA and the measurement of the acetyl-CoA by a coupled system of malate dehydrogenase and citrate synthase. The assay is specific as citrate synthase is specific for acetyl-CoA. The spectrophotometric assay is designed for either a dual or single beam instrument holding cuvettes of 1 cm light path. The fluorimetric procedure is designed for an instrument capable of handling 1.0 ml volumes. For the fluorimetric assay quinine sulfate in sulfuric acid is used as the reference standard; however, for each assay a standard curve of potassium acetate is run simultaneously. The fluorimetric procedure is usually less precise than the spectrophotometric assay. The standard error of the mean of triplicate tissue samples is 3–5%. The higher standard error of the mean in the fluorimetric procedure reflects the relatively greater significance of the acetate contaminations of the reagent cocktail at these very low concentrations of acetate.
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A simple, sensitive and accurate gas chromatographic method for the determination of acetate in plasma and urine is described. Following precipitation of proteins by perchloric acid and removal of potassium perchlorate by precipitation with KOH, acetic acid is quantitated by injection of the acidified sample on a highly polar column. A flame ionization detector is used. Propionic acid is added to the sample initially as internal standard. The sample volume required for the analysis is 0.5 ml. The standard curve is linear in the range from 0.01 to 10 mmol/l. Recovery of added acetate is complete, and the coefficient of variation is less than 4% even at low concentrations. The concentration of free acetate in plasma from 10, and urine from 4, healthy humans ranged from 0.04 to 0.07 and 0.09 to 0.24 mmol/l, respectively.
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A relatively simple gas Chromatographic technique is described which makes possible the identification and quantitation of free volatile fatty acids containing up to 6 carbon atoms in physiological fluids. Values are shown for the concentrations of these acids in the urine, serum, cerebrospinal fluid, and sweat of normal human subjects. The method can be valuable in the study of metabolic disorders involving the accumulation of one or more volatile fatty acids.
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The determination of formate, acetate and propionate as their pentafluorobenzyl esters by glass capillary gas—liquid chromatography has been studied. The separation of pentafluorobenzyl esters of acids is obtained with columns coated with PPSeb stationary phase. Quantitative results are given with samples containing ca. 5 nmol of acetyl and formyl groups.
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The products of a fermentation process may be few, as in the case of the classical yeast fermentation, or they may be tremendously diverse, which is typical of much bacterial fermentation. Therefore, the analysis of fermentation presents tasks ranging in magnitude from the relatively simple to the highly complex, involving the separation, identification, and quantitation of a broad spectrum of compounds. The development of column, paper, and thin-layer chromatography has had a major impact not only on the separation and identification of fermentation products but also on their quantitative determination. This chapter discusses the analysis of fermentation products, the general principles of fermentation balances, and the apparatus and techniques for carrying out fermentation studies. The chapter also discusses the procedures involved in the separation, identification and quantitative analysis of the various compounds likely to be encountered in fermentation processes. The identification and determination of volatile fatty acids by gas-liquid chromatography is also described.
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A method is described for the hydrolysis of proteins, amino acids and carbonhydrates for the liberation of N- and O-acetyl and formyl groups. The acetyl and formyl phenacyl esters are prepared by means of crown ether catalysis and determined by gas-liquid chromatography using the flame-ionization detector. Quantitative results are given with samples containing about 20 nmol of acetyl or formyl group. The method is also applicable to the determination of N- and O-propionyl groups.
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In four different strains of ascites tumors and in one solid hepatocarcinoma acetate was observed to constitute an end-product of aerobic metabolism of pyruvate and palmitate. According to enzymatic studies with soluble tumor extracts net formation of acetate can be ascribed to a deficiency of acetate thiokinase as opposed to acetyl-CoA deacylase which is efficiently active in tumor cells. The implications of the permanent loss of respiratory energy caused by enzymatic hydrolysis of the thioester bond of acetyl-CoA with regard to aerobic glycolysis of malignant tissues are discussed.
Extracorporal und sogar in unphysiologisch hohen Dosen per os oder intraperitoneal zugeführte Essig- und Citronensäure wird im Tierkörper in kurzer Zeit verwertet. Dabei tritt keine länger anhaltende Erhöhung der Gehalte dieser Verbindungen in Blut und Leber auf. 1 bzw. 2 Std nach der Verabreichung ist der Citrat- bzw. Acetatgehalt im Blut weitgehend normalisiert. Die Leber zeigt eine hohe Aufnahmekapacität für Acetat und Citrat. Dem im Blut durch intraperitoneales Überangebot hervorgerufenen sprunghaften Anstieg der Acetatwerte folgt kein entsprechender Kurvenverlauf in der Leber. Der festgestellte hohe Acetatumsatz der Leber wird diskutiert. Für die gelegentlich anzutreffende Ansicht, in der Nahrung zugeführte Essigsäure könne eine Belastung des Organismus bedeuten, finden sich auf Grund der vorgelegten Ergebnisse keine Anhaltspunkte.
Article
Flow injection determinations of acetate were carried out using immobilized acetate kinase, pyruvate kinase and lactic dehydrogenase with an amperometric method. Two acetate kinases from E. coli and B. stearothermophilus were tested. It was found that the immobilized acetate kinase from B. stearothermophilus was more stable than that from E. coli., but it is much more expensive and less available. Acetate kinase coupling at pH 7.4 using CPG aminopropyl and glutaraldehyde seems to be superior to other immobilization methods. A high immobilization yield can be obtained by immobilization of the three enzymes separately giving high conversions of all the three. Plots of current versus concentration show a useful operating range from 0.3 to 2 mM acetate with a linear response. The detection limit was 0.2 mM at a flow rate of 0.3 ml/min with 200 μl injections. The method is therefore well suited for monitoring of the level of acetate in fermentations with acetate as the carbon source.
Chapter
Mit der Entdeckung des Coenzym A im Jahr 1945 bestätigte sich von neuem die aus anderen Beispielen bekannte Tatsache, daß Vitamine als wesentliche Bausteine von Coenzymen in den geordneten Ablauf von Stoffwechselprozessen eingeschaltet sind. In diesem Falle war es die Pantothensäure, der man endlich einen ihrer vielfältigen Vitaminwirkung angemessenen Platz im Stoffwechselgetriebe zuweisen konnte. Die ungewöhnlich rasche und ergiebige Forschungstätigkeit auf dem Gebiet der Coenzym A-abhängigen Stoffwechselreaktionen ist zweifellos auf den hohen Stand der Methoden der Enzymologie zurückzuführen, in den das neue Coenzym hineingeboren wurde. Als Ergebnis liegen heute viele Dutzende in ihren Eigenschaften und ihrer Wirkungsweise gut bekannte neue Enzyme vor, die uns — von wenigen Ausnahmen abgesehen — ein nahezu lückenloses Bild von der beherrschenden Funktion des Coenzym A im Stoffwechsel vermitteln. Die systematische Abhandlung des Komplexes der Coenzym A-Enzyme darf nunmehr, nachdem die Ernte im wesentlichen eingebracht ist, in Angriff genommen werden.
Ab8tr. Comm. 4th int Acta phy8iol. scand. 50, suppl. 175, 97 Methoden der enzymatischen Analyse (in the Press)
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Lundquist, F. (1960). Acta phy8iol. scand. 50, suppl. 175, 97. Lundquist, F. (1961). Methoden der enzymatischen Analyse (in the Press). Ed. by Bergmeyer, H.-U. Heidelberg: Verlag Chemie.